JPH02503147A - Uniform protection test - Google Patents

Abstract

Improved homogenous diagnostic assay methods and labels for detecting an analyte in a medium when the analyte is a member of a specific binding pair. The methods and labels provide procedures for reducing background and increasing sensitivity. The binding partner of the analyte is labeled with a substance, the stability of which detectably changes whenever said analyte is bound as a member of the specific binding pair. In a closely related system, the analyte is labeled with a substance susceptible to differential degradation depending on whether or not the analyte is bound as a member of its specific binding pair. After incubation and selective degradation or chemical or biochemical alteration, the amount of analyte bound is detected by measuring either the stability change or the extent of degradation of the label. In a particular system, chemiluminescent acridinium ester labeled probes are used in a homogenous hybridization assay format for sensitively detecting the presence of complement any target polynucleotide sequences.

Description

[Detailed description of the invention] Uniform protection test background The present invention is in the field of diagnostic methods and techniques for detecting and quantifying trace amounts of organic compounds. It is.

More particularly, the present invention is directed to Concerning homogeneous diagnostic assays that differ in the stability of labels.

The present invention provides that the label is different in its composite or combined form compared to its non-compounded or uncombined form. This paper relates to the configuration of an environment for a diagnostic assay system that is then disassembled.

Background of the invention Diagnostic assays use labeled binding reagents to detect and localize biological substances; Or, it is a general analytical technique for quantitative analysis. The presence of the labeled reagent is then detected. It can be detected using various known methods. The present invention provides direct binding assays and competitive Sequential 5 saturation Applicable to all known diagnostic assay formats, including without limitation. one ingredient A typical diagnostic assay is a nucleic acid hybridization assay. Hybrid verification system A mercury nucleic acid (DNA or RNA) is a complementary mercury nucleic acid (DNA or RNA) under appropriate circumstances. It is based on hybridization or recombination. easily detected -mercury nucleic acid sequences by labeling the complementary probe nucleic acid with a label that can detecting the presence of a target polynucleotide sequence of interest in a test sample containing is possible.

Assay systems can broadly be heterogeneous or homogeneous. homogeneous). applied to the certification system. The term "heterogeneous" refers to the separation of labeled material from unlabeled material to be detected. Refers to a system for specific binding assay characterized by:

On the other hand, a homogeneous assay system is a homogeneous assay system that does not include any physical separation process. Because systems require the least number of handling steps, they are generally more convenient and easier to use than heterogeneous assay systems. Easy to use.

For example, the classic sandwich immunoassay uses immobilized antibodies together with test medium. Includes incubation polishing. If the antigen is present in the medium, the antibody Combine with. After incubation, unbound antigen is removed in a separation step. label After a second or co-incubation with the antibody solution, the bound antigen is immobilized. "sandwiched" between the labeled antibody and the labeled antibody. After the second separation step , the amount of labeled antibody can be measured as the amount of antigen in the medium. Two so time It takes.

In diagnostic assays, efforts in conventional techniques have focused on homogeneous assays and unbound target substances. The development of labels that can distinguish small differences in the amount of bound substances as opposed to One object of the present invention is that the label itself - e.g. When the assay system undergoes a detectable change in chemiluminescence capacity relative to Another object of the present invention is that the label is either in its bound or unbound form. from which the desired reaction can be identified and determined. It is an assay system that provides a ready-to-use means of quantifying

The present invention discloses an improved method for sensitive detection of analytes using a homogeneous assay format. The purpose is to The present invention also relates to the homogeneous method disclosed herein and other separation methods. The sensitivity of the assay encompasses separation to reduce non-specific background. The purpose is also to provide an improved method for increasing the degree of The principle of the invention is to In biochemistry, whenever a binding partner binds, the binding partner binds to it. The stability of the label changes when it binds to a substance, or I put it out. Moreover, the present invention uses a uniform diagnostic assay system to detect the difference in label stability. The present invention aims to disclose a method that can be adopted for sensitive detection of analytes. Yet another objective is to detect the presence of complementary target polynucleotide sequences with good sensitivity. The chemiluminescent, acridini westerla in the homogeneous diagnostic assay for the detection The purpose of this invention is to disclose the use of Pel DNA probes.

Description of prior art There are various homogeneity tests of varying complexity in the prior art. On some systems, e.g. For example, the label may contain a catalyst that can participate in a chemical reaction in the presence of other components, such as an enzyme. , coenzymes and cofactors.

In related systems, but in other cases, the label is a chemical or biochemical It is a substrate for reaction. These systems use specific, easily detectable substances, e.g. For example, monitor target reactions using glucose, lactate or alcohol; Measure. In other assay systems, the label has unique physical properties and can be directly is detectable. Example 1 of these labels includes metals, hemoglobin and Includes chlorophyll.

Other homogeneous assay systems are also based on enzyme-coupled specific binding reactions. So, the analyte becomes the ligand for the specific binding reaction.0 If the analyte is present, it is , a conjugate consisting of a specific binding partner and a label substance. undergoes a specific binding reaction. Other substances that simultaneously or subsequently interact with the label Add. The activity of the label depends on whether the conjugate of which the label is one component is in complex or non-conjugated form. It differs depending on the compound form. Such systems typically use enzymes as labeling reagents. and also using substrates that produce colorimetric, fluorometric or chemiluminescent endpoints. ing. Examples of homogeneous enzyme immunoassays include U.S. Pat. , No. 3817837 and No. 4190496. Fluorescent material ( Fluorescence quencher pairs involving the use of chromophores Other examples of one test are U.S. Pat. No. 4,199,559, U.S. Pat. Seen in No. 318707. However, in some systems the label is other than an enzyme. substances such as vitamins, NAD, FAD or biotin; However, these can be linked together. An example of this type is a U.S. patent No. 84383031. In these systems, the monitor reaction is based on the activity of the label. Based on structural changes that change gender.

Other homogeneous assay systems include polarized fluorescence techniques. Here, the analyte is a small molecule The amount of fluorescent conjugate competes for binding to high molecular weight binding partners. Fluorescence polarization is small The amount of analyte in solution changes as the molecule shifts from the surface of a larger molecule. It is possible to measure.

An example of this type of assay system is US Pat. No. 4,668,640.

Still other types of homogeneous assay systems include non-radiative energy transfer.

In these systems, when two molecules approach each other, light from one molecule is emitted by The absorption of is used as a monitor reaction.

Generally, these methods are described in two ways. In one method, the first molecule is chemiluminescent, and when excited, some of the electromagnetic energy generated It is transferred to the ``absorber'' molecule of Yuki 2, which should be nearby. M2 molecules give off energy A chemical in which some of the light is close to the absorber molecule when it absorbs and emits light at a different wavelength It exhibits a spectral shift proportional to the number of luminescent molecules.

In other types of non-radiative energy assay systems, the first molecule is a fluorophore and the becomes an ``absorber'' of light. The presence of two fluorescent molecules close to each other; "absorber" and exhibits a spectral shift proportional to the number of "emitter" molecules.

Another type of double probe assay system is found in U.S. Pat. No. 4,670,379. It will be done. The fourth probe is labeled with a catalyst and the second is apo-luminescent (apo-luminescent). luminescer). Both probes target nucleic acid sequence Target areas adjacent to . Once both probes have hybridized to the target , add substrate. The catalyst converts the substrate into converted radicals, which in turn generate a second glow. Change the apoluminescent material on the board to a luminescer. This is a hybrid Occurs only when grid conversion occurs. Using these principles, a common analyte can be An assay based on two labeled substances that bind to I has been developed.

A specific example of this type of energy transfer verification system is Published Elazar et al. European Patent Application No. No. 85105130.0 (publication number 0159719), and the target gene product discloses the use of two monomercuric nucleic acid probes that complement identical or opposing strands of quality. Ru. Each probe is a part that can generate a signal only when two labels come together. Labeled. Unlike the present invention, eraser et al. double hybrid or Includes multi-hybrid formation and detection. Similarly, January 2, 1983 European patent publication of Heifer et al. issued on the 6th No. 82303699.1 (Publication No. 0070685) and related European Patent Application No. 823 of Morrison et al. No. 03700.7 (publication number 0070686) uses two luminescent probes. A uniform verification system is disclosed. At least one of the optical labels is an absorber/emitter. When two probes hybridize to a target, two lights are generated. Energy transfer occurs between the bells. @2 light emission is hybrid Detect formation of a cord. This type of antigen assay is disclosed in Morrison et al., supra.

Most biological substances cannot be easily and directly detected at any reasonable sensitivity level. , some type of binding reaction is required. As is clear from the above, Prior art cannot significantly distinguish between bound and unbound labels. Such a test is highly Detection of analytes present in concentration, e.g. monitoring of various drugs in blood and urine It is useful.

In the field of clinical diagnostics, the ability to alter the stability of the label of two binding partners, e.g. For example, binding or selective removal of a label in the unbound form is based on destruction. Requires direct detection homogeneity test. hirshefie yield ld) Fluorescence Ba ckgground Discriffiination by Preblea Ching), Journal of Histochemistry and Cytochemistry Tory (J, Histochemistry and Cytochemis try), 27/1.96-101 (1979) is a label molecule or at least Somewhat related electronic light also involves photochemical bleaching, which can destroy their fluorescence. However, the present invention is not applicable to photochemical bleaching or diagnostic assays. or suggest. The present invention has several orders of magnitude better sensitivity than the conventional technology. and meets current requirements in diagnostic assays. The sensitivity range of the conventional technology is 10 The present invention is sensitive in the 10-" molar range, although not much better than the 10-" molar range.

Outline of the invention In summary, the present invention consists of diagnostic assay controls and methods. The method uses an analyte in a medium. and the analyte is part of a specific binding pair. Contains the analyte When a medium that seems to be combined with a binding partner, the label substance bound to the binding partner becomes a detectable change or difference in stability each time an analyte binds to the specific binding partner undergoes decomposition. In a specific example, the -terminal strand nucleic acid probe is substantially Any desired moiety or I; is modified to include a label in place. plow The stability of Bralabel depends on whether the target nucleic acid sequence hybridizes to the probe or not. or are susceptible to different degradation.

In particular, the invention provides that the label on the bound probe is stabilized with respect to the unbound probe. It consists of a detection system. This stabilization is an intercalation facilitated by DNA insertion compounds, including acridinium esters, are particularly , but not exclusively, are suitable for use in the assay system of the present invention. D It is known that NA inserts bind non-covalently to double-stranded DNA, and It is a characteristic flat molecule that can be inserted between the base pairs of the double helix of A. The inventors Acridinium esters preferentially insert into regions rich in adenine and thymidine base pairs. I have proof that it will be accepted.

In the following, the invention will be described with particular reference to probes labeled with acridinium esters. However, the present invention provides for differential degradation when a label is attached to a component conjugate. Also, use uniform labels and assay formats that are sensitive to changes in stability. should be understood as intended. As further described herein , other suitable labeling compounds include amines present in nucleic acids, especially non-hybridized Includes substances that are destabilized by amines near the label on the probe. Sara Labels that can interact with the hydrophobic environment, e.g. by insertion between base pairs. Quality can also be used.

The present invention generally provides for selective labeling of labels when comparing bound and unbound conjugate forms. Applicable to any system involving substantial decomposition. In addition, any minute Because the assay system is relatively simple and does not involve separation or cleaning steps, it is faster and less expensive. Stability between bound and unbound conjugate forms of the label If there is a large difference, any residual or unbound label conjugate will be destroyed; This makes the system even more sensitive than traditional systems. . Finally, the system is very flexible and can be used for both simple and narrow separation methods. It can also be used in combination with other methods to achieve very low background noise. 0 The present invention provides probes for the detection of infectious diseases, genetic and viral diseases, and cancer diagnosis. Useful for diagnosis.

Brief description of the drawing Figure 1 shows HPLC analysis of acridinium ester-label probe Figure 2 shows an example 2 is a graph showing the results of No. 2.

Figures 3 to 5 are graphs showing the results of Example 3.

FIG. 6 is a graph showing the results of Example 4.

FIG. 7 is a graph showing the results of Example 5.

FIG. 8 is a graph showing the results of Example 6.

DETAILED DESCRIPTION OF THE BEST MODE AND PREFERRED EMBODIMENTS The following definitions are used in this description: .

1. Acridinium ester: has a quaternary nitrogen center and is derivatized at the 9-position. derivatives of acridine that produce unstable phenyl ester groups, specifically 4 -(2-succinimidyloxycarbonylethyl)phenyl-1O-methyla Chrydiainium 9-carboxylate fluorosulfate: 2, Acrylic Part of the secondary general formula of dinium esters: R8-alkyl, alkenyl, ary substituted alkyl, substituted alkenyl, substituted aryl, alkoxy, arylox Stripe I; is absent in the case of X-halogen.

R1-H, alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl, Substituted aryl, alkoxy, aryloxy, only if X-N.

R, -H, amino, hydroxy, thiol, halogen, nitro, amino, amide , acetyl, alkyl, alkenyl, aryl, substituted acetyl, substituted alkyl, Substituted alkenyl, substituted aryl, alkoxy, aryloxy.

R4-Alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl, substituted Aryl.

X-O%N%S1 halogen, substituted phosphorus, substituted sulfur, substituted boron or substituted arsenic.

Y-0% S or NH.

R1 and/or R2 and/or R3 and/or R4 chemical conjugation It has a reactive site that allows for

3. Analytes - including but not limited to antigens and antibodies against them, haptics Ten and its antibodies, hormones, medicines, metabolites, vitamins, coenzymes and and its binding partners, including receptors, polynucleotides, oligonucleotides, polynucleotides, Cleotide or oligonucleotide hybrids and antibodies against them and binding substances, polynucleotides or oligonucleotides and their conjugates. Hybrid-forming polynucleotides or oligonucleotides, metals and the ability to bind with one or more specific binding partners, including chelating agents for any substance that is.

4. Binding partner - any molecule or substance capable of a specific binding reaction with the analyte quality.

5. Double-stranded structure: A double-stranded structure formed by the annealing of two complementary-terminal nucleic acid molecules. Main chain complex.

6. Hybridization between two complementary-end strand DNA or RNA molecules! : is DN Formation of a stable double-stranded structure between A and complementary RNA. Double-stranded structure formation is based on complementary bases pair-specific, thereby causing hybridization; inheritance of specific genes The code is played.

7. Binding: Conditions under which binding interactions occur between a molecule and its specific binding partner.

8. Stable: against chemical or biochemical decay, reaction, decomposition, substitution or modification. resistant.

9. Stability: Chemical resistance to biochemical decay, reaction, decomposition, substitution or modification. Tolerance of substances.

It is known that acridinium esters exist in equilibrium with the corresponding base in solution. It is being At high pH, base formation is dominant and quaternary nitrogen species are present at low p It is reformed with H. Chemiluminescence reactions occur with bases, especially hydroxy acids containing hydrogen peroxide. It is also known that the addition of an aqueous sodium chloride solution can have an effect. chemiluminescence includes attack by hydroperoxide ions on acridinium species. and generates electronically excited N-methylacridone. Overall, Weeks (Weeks) et al. Lysinium Esters”, Clinical Chemistry (C1in, Chew, ), 2918.1474-1479 (1983). The reaction is diagrammed below. do.

Acridinium ester reaction diagram The invention can be implemented as follows. First, the combination for the test to be performed Selecting a binding partner consisting of a substance and one or more binding partners. these Pairs are antigens and their antibodies; haptens and their antibodies: hormones, drugs, and drugs! f product, Its binding partners include vitamins, coenzymes and receptors: polynucleotides, Oligonucleotides and polynucleotides are oligonucleotides. Brids and antibodies and their binding substances; polynucleotides or oligonucleotides polynucleotide or oligonucleotide that can hybridize with gold; genus and its chelators.

Second, select the assay format to be used. These include direct binding tests, competitive Sequential saturation method ds) and the Sandinch test. .

Third, select a label for the assay to be performed. This can be directly or indirectly compared to For colorimetric, fluorometric, chemiluminescent or bioluminescent methods Any label that is more detectable may be used.

In addition, the label modifies its ability to be detected by chemical or biological It has the property of being chemically degraded, and such degradation occurs between the labeled binding partner and its of the compound and other binding partners that may be involved in the reaction. This is possible under conditions that do not adversely affect the bond between. Preferred labels are acid, After irradiation with a selective oxidizing agent such as a peroxidate or an enzyme, the base or It is something that affects its ability to be detected.

Fourth, labeling for chemical or biological degradation using chemical methods known to the optical industry. sensitivity to the interaction between a labeled binding partner and its specific binding substance(s). A label is attached to the binding material at the site to be modified. In some cases, multiple Test different sites for label binding and use the site that gives the most specific degradation. can be done.

Fifth, optimal detection of labeled binding partners with or without their binding substances. Maximize the use of chemical or biological degrading conditions that confer identification.

Finally, test the proficiency of the certification system using a pre-selected certification format and generally : a. Incubate; b6 selectively degrades, Do you want to detect C0? a. Simultaneous incubation and selective degradation; b. Detection. The analytes were labeled with chills to be detected qualitatively and quantitatively using a process of Oligonucleotide probes generate specific polynucleotides via hybridization. Particularly useful for detecting nucleotide sequences. acridinium ester, 19 US patent application filed on September 21, 1987! (not yet numbered), “For nucleotide probes” by Arnold, et al. DNA probes and/or or at many different sites on mixed nucleotide/non-nucleotide polymers You may let them. This includes nucleotide bases, phosphate backbones, and oligonucleotides. 3' and 5° ends of leotides and mixed nucleotides/non-nucleotides Includes the ability to label a single non-nucleotide monomer unit of a polymer.

Such acridinium ester-labeled probes are If the probes to which they are attached are free in solution, In some cases, they may exhibit significantly different chemical stabilities. This different stability makes the probe acridinium ester, nucleo near the acridinium ester label Position of tide residue and stable hybridization with target polynucleotide dependent on the presence of other probe molecules to form. A graphical representation is shown below.

Contributing to the different stabilities of acridinium esters in DNA probe molecules In the method of measuring factors, non-hybridized globes (nucleotides and Free amines on bases (both mixed nucleotides/non-nucleotides) It was found that acridinium ester becomes unstable in a potassium atmosphere.

At the same time, if the acridinium ester is adjacent to the end of the probe, When hybridization occurs, it Therefore, it may be unstable to alkali. The probe is a specific polynucleotide ( When hybridizing to a sequence (binding agent), the probe amine It is related to the base that pairs with otide, and its ability is limited, especially under alkaline conditions. destabilizes acridinium esters; At the same time, hybrid duplex hybrid duplex, especially the adenine/thymidine base pair density By inserting the acridinium ester into the position, the acridinium ester is further stabilized against degradation. It turns out that it does. Many of the Other insertion sites were also found to provide good variable hydrolysis.

There are several embodiments in which the present invention can be applied to hybridization. . These are not intended to be limiting.

1. Acridinium ester at the center of the probe and adenine/thymidine salt It is attached to the adjacent part of the base pair. A preferred method is the US patent application filed September 21, 1987. National patent ratio Wit (not yet numbered), Arnold et al. , et al.), “Non-nucleotide binding reagents for nucleotide probes” Acridinium esters are combined with non-nucleotide monomers as described in attached to the unit, i.e. immediately adjacent to the target polynucleotide sequence A nucleotide monomer that is complementary to a nucleotide is inserted into a single unit and attached. It is to make it possible. Such an arrangement allows the nucleotides at both ends of the acridinium ester to It serves to limit the amine of the otide base and provide a site for insertion.

2. Attaching the acridinium ester to either the 3' or 5' end of the glove and the amino acid contributed by the target polynucleotide with the second probe. to limit the action in the vicinity of the probe and hybridize the vicinity of the first probe. The second The probe creates a site rich in <A/T base pairs based on duplex formation. If so, that is also desirable. The two and Eko's double probes Sandwich systems rely on the formation of two duplexes instead of just one. Even if the probe is very short, i.e. about 5-15 nucleotides in length Sensitive detection of minor base pair changes that can be identified by probes provide a method to do so.

3.7 Adding a clidinium ester to a polynucleotide that is not the desired target polynucleotide It is attached at or near the site of mismatch with the nucleotide sequence. this As such, the duplex region around the mismatch site is significantly destabilized and clidinium ester (if it is attached at or near this site) ) is more susceptible to degradation, so only one nucleotide Discrimination between polynucleotide sequences that differ can also be achieved.

One preferred embodiment of the above three formats is hybridization A different hydrolysis step is carried out at the same temperature as the incubation step, typically 50-70°C. That's true.

Another embodiment is to carry out another different hydrolysis step at room temperature. This is l Using a pH range of 0 to 11, hybridized and non-hybridized acridinium yielding greater differences in hydrolysis rates between muester-labeled probes. .

Under normal circumstances, such short gloves should be used under suitable hybridization conditions. It has the ability to identify a single mismatch. However, they are They are not routinely used as they are not significantly specific for nucleotide sites. that is, the short probe is not used in many complex DNA may hybridize multiple specific sites contained within the A or RNA sequence. No, two different probes hybridize immediately adjacent polynucleotide sites. Under the condition that it has to be and can be identified. Therefore, the advantage of short probe identification is that both probes can be used while maintaining site specificity. .

Experimental methods and materials The following examples are illustrative and not limiting; these examples are It is based on generally available data and the best explanation of the phenomenon. other Factors and methods may emerge with further research. The present invention Described in detail with illustrations and examples using the clidinium ester label However, certain variations and modifications may be made within the scope of the claims, and other label systems may also be used. It may also be used within the scope of the claims.

Example 1 Assembly of acridinium ester-labeled probes deoxyoligonucleotides The 5°-terminus inside the probe, along the polyline a chain, was selected in advance. Amino acids located at any of the positions or attached to one nucleotide base linker arm (i.e., the primary arm labeled with the acridinium ester) It was synthesized so that it had a compound (terminated at min).

(i) The following compound was used to attach the 51-amine linker-arm to the probe: there was: This compound has previously been referred to as a terminal amine linker-arm reagent. child The reagent was synthesized as follows: 6-aminohexanol in anhydrous ethyl acetate. The 0-reacted purified product reacted with S-dityl trialolothioacetate is petroleum ether. precipitated in a 10% pyridine solution and treated for 10 minutes with a mixture of lO% pyridine in water to form a The refluoroacetyl was hydrolyzed and evaporated to dryness to a gum form. Then, this compound The material was purified according to standard protocols described in the literature. (hosphitylate) L, the desired compound, i.e. the terminal amine linker --Arm reagent (1) was obtained.

A probe with a 5°-amine linker-arm was synthesized as follows. app Ride Biosystems Inc.3 (Applied B1o system+ns, Inc,) model-38OA DNA synthesizer according to standard phosphoramidite chemistry using Then, obtain a probe of the desired nucleotide sequence, and probe from the 3° end to the 5° end. was formed. After the desired sequence is completed, the amine linker-arm can be attached to another host. Just as foramicytonucleosides couple, terminal amine phosphorus Automatic coupling to the 5'-hydroxyl group of the probe using Kerr-arm reagents I nged. using standard protocols, then cleaving the probe from the solid support; Deprotection, polyacrylamide gel electrophoresis followed by Sephadex (Sephadex) phadex) G-25 chromatography.

The following probe was synthesized and purified according to this procedure: 5-face 2- (CI!2) 6-GC’l’CGTTGC (2) AC cost MCC gift CAT-3” (N Hx-C CHx)a indicates amine linker arm) (■) An internal amine linker is used to incorporate the amine linker arm into the interior of the probe. Arm reagent, Type I or Type 2, is covered by a U.S. patent filed September 21, 1987. Application No. 099050, Arnold et al. Nucleotide Binding Reagents were used as described in ``Nucleotide Binding Reagents''. Again, set the probe to standard Synthesized using standard phosphoramicite chemistry and analyzed by polyacrylamide gel electrophoresis and It was purified using Sephadex G25 chromatography.

The following probes were synthesized according to this procedure: 1) 30mar complementary for 165 subunit rRNA from is an internal amine linker-arm, type I, with an adenine residue at position 18 in the sequence. Replace = 2,) Chlamydia patrakomate (su(Chlamydia t rachognatis) or internal amine linker-arm, type l, in sequence replacing the adenine residue at position 21 of or inserted between residues 21 and 22: (m) A nucleotide base with an amine linker-arm is attached to the probe as follows: Inserted. Synthesize template and primer oligonucleotides with the following sequences: Ta: Mold 3°-GCA ATG AGCCTA CGG GTTTAT AGCGG -5゜ Primer 5°-CGT TACTCG GAT GCCCAAAT-3' (Primers and elongated primers are 165 subunits of C. trachomatis. It is complementary to ) Primer extension using Klenow 7-ragment BSA The map is omitted from the Proceed as described by Maniatis (ref,), Amine linker-arm modified base amino (12) dUTP (Calbiochem, CA (Calbiochem, California) was inserted.

Therefore, the sequence of the obtained oligomer is: CGT TACTCG GA T GCCCAA ATA (amino-12-U)CG CC.

Purification of the primer extension complex was carried out using NENSORB-20 cartridges (DuPont). (]) u　P　ont) and followed the manufacturer's recommendations. ( The primer extension reactants were treated with NENSORB reagent A900 reagent before loading onto the column. The total amount of medicine was 9002g. The purified oligomer was eluted with 50% methanol and then washed. Remove with speed-vac.

B, Labeling of amino linker-arm probes using acridinium esters. oxidation and purification A 25mM stock solution of acridinium ester was prepared in distilled DMSO. Ta. Desired amount of probe (various labeled probes in Section A above) ) was evaporated to dryness in a 1.5 ml+ conical polypropylene tube.

The cocktail was made up by adding the following ingredients in the order listed: 3μg water 1μ (l IM HEPES (pH 8,0) 4pQ DMSO (distilled) Stir 25mM acridinium ester mixture in 2μQ DMSO (distilled) Then, spin in a microcentrifuge for 2 seconds to concentrate the sample at the bottom of the tube. The cells were incubated for 20 minutes at 37°C. Then, add the following ingredients, Add to the reaction cocktail in the order listed: 3.0 μ(l) 25+nM acridinium ester in DMSO (distilled) 1.5 μg water Stir the 0.5μQ IM HEPES (pH 8,0) cocktail again and The mixture was incubated for an additional 20 minutes at 37°C. Unreacted labels are 5 Using a two-fold excess of glue, 0.1M HEPES (pH 8,0), 50% DMSO Quench by adding 0.125M lysine and ink for 5 minutes at room temperature. It was incubated.

Next, Akridani 9 Muestermo It was purified using the method of 20 μg of the quenched reaction mixture was added with 3M as carrier. Na0Ac (pH 5,0) 30 pn, water 20tt (l and glycogen 5 μg was added (the glycogen was pre-washed to remove any nuclease activity). The sample was briefly stirred and 640 μg of absolute ethanol was added. The sample can be easily stirred. Mix and incubate on ice for 5-10 minutes, then place in a microcentrifuge. The mixture was centrifuged at 15,000 rpII+ for 5 minutes. Carefully remove supernatant and Pellet) to 0. lhi　Na0Ac (pH5-0) 20μm2.0.1%S Dissolve it again in DS. The sample was then subjected to high performance liquid chromatography as described below. Purified by Laffey (HPLC).

(i) The AE-labeled probe with 5' and internal amine linker-arms is Purified as: The redissolved pellet was loaded onto an IBM 9533 HPLC system. Mounting I: Nucleogen-DEAE60-inject onto ion exchange HPLC column. Ta. All buffers are manufactured by Fisher Scientific (F1she). HPLC grade water from rScientific), acetonitrile (CHI CN) and sodium acetate (NaOAc), reagent grade glacial acetic acid (HOAc ) and LiCQ. All buffers were prepared with a 0.45 μm pore size prior to use. was filtered through a nylon-66 filter. Specify the sample as shown in Figure 1 (in this case). In this case, the probe contains the 5' -amine linker-arm). 5! ! Immediately after the trial, 1 55 μg of 0% 5D was added to each tube, followed by stirring each tube (this , to prevent the acridinium ester-labeled probe from sticking to the test tube wall. Nishit;). Take a 0.5 μg portion from fractions 21 to 42 and transfer to a 12×75 200 pff of water in a mm test tube (using a separate pipette tip for each part) , which avoided the carryover problem). Next, check the chemiluminescence of each part. , using the following automatic injection and reading sequences: Measured at thold C1inC11nilu: 0.25N HNOs 200 p Q, O, 1% H, O, (7) injection; 1 second later; l N NaOH 200 μg injection: Chemiluminescence output reading for 10 seconds.

Fractions 29-33 were then EtO)I precipitated as follows: each fraction Add 5μg of glycogen to a comb and stir, add 1ml of EtOH and stir. Incubate on ice for 5-10 min and in a microcentrifuge for 150 min. Centrifugation was performed at 00 rpm for 5 minutes. Carefully remove each supernatant and pellet Dissolve it again in 0.1M Na0Ac20μQs pH5, O01% SDS, and Pool the traction and t2.

In this way, a highly purified 7clidinium ester-labeled probe was obtained. mosquito The specific activity of such a probe typically ranges from 5 to 1 pmol of oligomer. 10xlO' chemiluminescence light number (Bertholt-Krinirmatt). Ta.

(ii) AE-labeling with amine linker-arm modified base (amino-12-U) Probes were generally purified as described above with the following exceptions: Vydac. (Vydac) A C4 reverse phase column was used; Buffer A was 0, 1M acetic acid trichloride. Ethylammonium (Applied Biosystems, Inc., California, Hoster City (-(F　C1ty)), Buffer Solution B was cHsCN; the labeled probe was added at a flow rate of 1 ml/min for 25 min. Elute as a hybrid using a linear gradient of lO to 15% solvent B in min. Ta. The main chemiluminescence peaks were then identified and post-treated as described above for I ;.

Generally, the above discussion involves synthesizing probes with acridinium esters. , describes how to label. In particular embodiments, the probe and internal labeling, as well as labeling with nucleotide bases.

Example 2 Hybridization probes internally labeled with acridinium ester and non-hybridized Stability of hybridized probes at pH 6 Chlamydia trachymatis (C Internal labeling 3 specific for Hlamydia tracho + natis) The 3mar probe was prepared as described above (adenine substitution, type I linker). - arm). The probe is linked to its target rRNA (in this case was hybridized with Chlamydia trachymatis).

hybridization mixture 2μm 2AE-globe (0,5 pmol) 0,:3μm 4% (weight: volume )SDS5.8μm21MPB%pH5 4,4 μQ C. trachymatis rRNA (2 μg) or control 4.4μQ water The hybridization and control mixtures were incubated at 60°C for 40 minutes. and analyzed it. 01 μQ of the hybrid or control mixture was added containing 2% HAP. 0.14M PB% pH 6.8° 200 μQ. Obtained I; Stir each mixture for 5 seconds, incubate at 60°C for 5 minutes, stir for 20 seconds. and then 1soo.

Centrifuge for 30 seconds in a microcentrifuge at rpm. Remove the supernatant and keep it? FL, 0. Add 14M FB, pH 6, 8, 200/712 to HAPpet L/7 and mix. The mixture was stirred for 10 seconds and then centrifuged for 30 seconds in a microcentrifuge at 15,000 rpm. I was concerned. Remove the supernatant, save and pellet in 0.14M PB%p) (6, Resuspend at 8,200 μg. 50 μg of resuspended HAP and 2 supernatants each were analyzed for chemiluminescence as described below and the percent hive Percent chemiluminescence signal associated with lidded globe, i.e. HAP I calculated it.

of hybridized and nonhybridized probes (i.e., control). Stability was tested at pH 6 according to the following procedure.

Stability test mixture 12.3 μg hybrid or control 50pQ IM PB from above, pH6 2,5μQ 4% 5DS 65μg water Gently stir these mixtures and immediately remove 5 μg aliquots, as described below. The chemiluminescence was thoroughly analyzed. Incubate the remainder of the mixture at 60°C. At each time point (see below), remove 5 μQ aliquots and immediately apply chemical lubrication. I analyzed the minerals on the island.

The chemiluminescence of each sample was determined by dipping a portion of the sample into a 12 x 75 mm test tube with 20 ml of water. Clinilumat (0.0 μg) was added to Clinilumat (0 μg).

25N HNOI 200/J (1,0,1% H80, automatically injected, 1 second delay) automatic injection of 2MFB potassium 200μ (i, pH 13-2, 10 seconds chemistry) Measured by measuring chemiluminescence (luminescence reading) Established.

result: 1. Percent hybridization (HAP analysis) hybrid - 96% Control - 1.3% (non-specific binding) 2. Stability time history 'See #g2 diagram These results demonstrate that the hybridized probes are not subject to degradation and subsequent chemiluminescence. Although well protected against loss of chemiluminescence (half-life of loss of chemiluminescence is equal to 3370 minutes), the non-nobridized probe is decomposed and chemiluminescent. very sensitive to loss of shows that the difference between hybridized and non-hybridized is equal to 115 times. vinegar.

imparting different stabilities between hybridized and non-hybridized probes and Demonstrate the ability to localize target DNA sequences by measuring It is something.

Example 3 Hybridization probes internally labeled with acridinium ester and non-hybridized Stability of hybridized probes at pH 9 Internally labeled probes (described in Example 1) 1: all three internally labeled 33mer probes tested) with their targets. Hybridization with get rRNA (in this case Chlamydia trachomanis) and analyzed for percent hybridization as described in Example 2. Analysis I:.

of hybridized and nonhybridized probes (i.e., control). Stability was tested at pH 9 according to the following protocol.

Esu 1 ward 1 eup 1 boat 5μQ Hybrid from above or control 45Pρ 0, 2M sodium borate The mixture was then incubated and sampled, Example 2 Chemiluminescence was analyzed as described in .

result: 1. Percent hybridization (HAP analysis) a, adenine substitution, tie Plinker-arm hybrid-95% b, insert, type l linker-arm Hybrid - 98% Control - 0,3% C0 insertion, type 2 linker-arm Hybrid - 98% Control - 0,2% 2. Stability time history See Figures 3-5. Figure 3 shows adenine substitution, type I linker arm. The M4 diagram is the graph for the inserted, type 1 linker-arm. Yes; Figure 5 is a graph for inserted, type 2 linker-arms.

As in Example 2, hybridized probes are protected from degradation, but non-hybridized probes are Hybridized probes are not protected. This is all about internally labeled probes. This applies to the three types.

In this example, sodium borate at high pH while still retaining different resolution characteristics between the lobe and the unhybridized probe. It is shown to accelerate this process (compared to Example 2).

Example 4 Labeled with acridinium ester as a hybrid with adjacent probes Stability of probe ends and non-hybrid at pH 6 This example uses the 5° end of the AE-labeled glove (see below) used in this example. It hybridizes to the adjacent E. coli RNA, and All in the target rRNA in close proximity to the acridinium ester label by leading to “uncapping” of the amine (using standard phosphoramidite chemistry) prepared) sequence 5″-CCG GACCGCTGG CAA CAA Including use of AGG ATA AGG GTT GC-3″ probe .

Labeled with acridinium ester I; probe end (prepared as described in Example 1) ) (hereinafter referred to as AE-globe) and the adjacent probe described above in the following procedure. Therefore, it was hybridized.

Hybridization E-scene mixture Control mixture 1pQ AE-probe LμL2AE -glove (, 25 picmol) (,25 pmol) 2pQ E. coli rRNA 5.4p (l water (2μm2g) 4.3μQ adjacent probe 5.8μIMFB% pH5 (12 pcom ) 0.3pQ4%SDS O,3pQ4%5DS7.5μρ 1M% pH5 The hybridization and control mixtures were incubated at 60°C for 40 minutes. hydroxyapatite as described in Example 2. -cent hybridization was analyzed.

Hybridized and unhybridized probes with adjacent probes ( That is, the stability of the control) was described in Example 2 and accurately tested at pH 6. I tried it.

result: 1. Percent hybridization (HAP analysis) Hybrid-93.5 % Control - 2.7% (non-specific binding) 2. Stability time history See Figure 6 As in Example 2, the hybridized AE-probe (in this case also hybridized neighboring probes) were protected from degradation, whereas non-hybridized probes were protected from degradation. The hydrogenated probe was not protected. The protection was as good as in Example 2.

This is because it relies on two hybridization calendars instead of one. .

Example 5 Labeling with acridinium ester as a hybrid with “flanking” probes Stability of probe ends and non-neutral hybrids at pH 9 Hybridization was performed exactly as described in Example 4. Shown below Adjacent probes were prepared as described in Example 2, except for the composition of the stability test mixture. Hybridized and unhybridized probes (i.e. , control) was precisely determined at pH 9.

5pg hybrid or control 50pQ 0.2M Sodium borate, pH 9.0 (also hybridization Hybridized AE-probes with adjacent probes (with hybridized probes) are again protected from degradation. protected, but the non/q-bridded probe was not protected and I; In Example 3 As a general rule, sodium borate at high pH /- to accelerate the process while still retaining different degradation characteristics between the hybridized probes. t;. For a graphical representation of these results, see Figure 7. Example 6 Acridinium ester-labeled probe ends as hybrids and Stability of Non-Hybrid at pH 6 The target rRNA (C. coli in this case) was hybridized.

Hybridization mixture Control mixture 1pQ AE-probe 　　　　　1ixQAE-Probe (,25 pmol)　　　　　　( , 25 pmol) 2p (1 E. coli rRNA 5.4p (1 water (2 μg) 5.8μQ IM PH, pH55, 8μm21MPB% pH50, 3μQ4 %SDS O, 3μQ4%5DS3.4μC water The hybridization and control mixtures were incubated at 60°C for 40 minutes. and then permeate using hydroxyapatite as described in Example 2. An analysis of the centhyperidic acid 2 was carried out.

of hybridized and nonhybridized probes (i.e., control). Stability was precisely tested at pH 6 as described in Example 2.

result: 1. Percent High Predye Scene Hybrid - 94% Control - 2.7% (non-specific binding) 2. Stability time history See Figure 8.

For non-hybridized probes, the difference in degradation was not as large as in the previous examples. However, again, it was significantly degraded compared to the hybridized probe. child This means that only a portion of the amine is “uncapped” in close proximity to the acridinium ester label. ” because it was done. In fact, this further increases the presence of hybridized neighboring probes. Distinguish between the hybridized end and the labeled probe in the presence and absence of Demonstrate ability to differentiate.

Example 7 Acridinium ester-labeled nucleotide base/9-bridded probe Stability of hybridized and unhybridized probes in p)(7,6) The nucleotide base (amino-12 -U) labeled probe to its target rRNA according to the following procedure. (In the second case, hybridize with C. trachomanais).

hybridizer 1 mixture 0.1M lithium succinate, pH 5, 41O% lithium lauryl oxide 2 μg 1.3 pmol AE-probe Total capacity -30μQ Hybridization and control mixtures were heated to 80°C for 5 minutes, followed by 60°C. and incubated for 60 minutes. The obtained solution was mixed with 0.1M lithium succinate, Diluted to 300 μg each with pH 5, 4, 1O% lithium lauryl WL oxide and carried out. Percent hybridization using hydroxyapatite as described in Example 2. Analyze the equation:.

of hybridized and nonhybridized probes (i.e., control). The stability was tested with several identically prepared samples according to the following protocol. Tested on H7.6.

Heaven! ``Madashi ni Tsuyoshi'' Tatsuo 15 μg hybrid or control from above 100 μm 1 0.2% tetrafos Sodium oxalate, pH 7, 6, 5% Triton X-100 These mixtures were incubated at 60°C and samples were taken at various times. Then, automatic injection of 200μg of 0.1M H2O2, IN NaOH with a 1 second delay Using automatic injection of 200 μg and 5 seconds chemiluminescence reading. Gen-Probe Leader Luminometer Chemiluminescence was measured using ether). From these data, hydration Degradation rates were determined by regression analysis.

result: 1. Percent Hypurity Analysis 1 (HAP Analysis) Hybrid-5368 % Control - 0,5% 2. Stability Half-life of ester hydrolysis (min) Half-life ratio Hybrid Control (Hybrid/Control) 13.6 1.3 10.5 As in the previous examples, these data are The probe is protected from degradation, but the unhybridized probe (in this case (with a label attached to the base of the probe) indicates that it is not protected. This is with AR Principles by which multiple sites for protection can be used in the uniform protection assay described herein shows.

Example 8 r　N　, A and DNA targets are used together to target at one site of various array dimers. Internally labeled with clidinium ester; hybridized and non-hybridized Stability of the probe at pH 7, 6 Various sequence dimer sites (see below) synthesize a probe containing an internal linker-arm, type "x", inserted into Labeled with AE and purified as described in Example 1. The arrangement of these probes The columns and linker-arm positions are as follows.

Probe No., Sequence linker-arm position (#)l 5°-GCT CGC GCGGA CTT#AAA CCA ACA T-3’2 5’-A to G Ding CG GTCT # Ding T CTCTCCTTT CGT CTA CG-3'35°-CAA TCG TCG AAA CCA TT#G CTCCGT TCG A-3' 4 5’-CCG CTA#CCCGGT ACG TT-3″5 　　　　　　　　5’-TTG　CCCACA　CCG　　A#CG　GC G-3'6 5'-TTG CCCACA CCG C#CG GCG- The 3'80℃ incubation step is omitted, and the target nucleic acid used is as follows. These probes were hybridized as described in Example 7, except that Izushi t2.

Probe # Target nucleic acid l E. coli rRNA (7) lug2 and 3 Q. - Q. trachomatis rRNA l, ug 4. N. gondrrhoeae (N, gondrrhoeae) r RN 12g of A 5 and 6 1.2 picomole of accurately synthesized NA complement as described in Example 7. In particular, the stability of hybridized and non-hybridized probes is determined by adjusting the pH. Tested at 7 and 6.

result: picture! i half-life ratio 1 41.2 0.94 43.72 24.2 0.71 34.53 40.7 0.96 42.4 4 15.7 1.2 13.15 11.1 1.0 11.16 22.6 0.74 30.5 These data indicate that the linker-arm (and thus the AE) has a wide range of sequences. It can be inserted into one site of dimer, and even in hybridized form, it can be prevented from ester hydrolysis. In addition, it indicates that it is substantially protected.

Additionally, this example shows that when used in these homogeneous assay formats, the DNA The target provides good protection for the hybridized AE-probe. In addition, this example shows that the long (9 atoms) linker arm used here has a wide range of links. Linker-arm lengths that can be used in the HPA formats described herein. Substantially the same as the other linker-arms cited earlier in this patent which established that It is also shown that differential hydrolysis ratios such as

Example 9 Perfect; hybridized with matching target and single mismatched target Safety of internally labeled AE-probe hybridized with target at pH 7.6 qualitative inserted between residues 6 and 7; internal amine linker-arm, type "x" The 24mar complementary to the 16s subunit of rRNA from E. coli with The probe was synthesized. The probe was then acrylated as described in Example 1. It was labeled with dinium ester and purified. The sequence is as follows (#- linker-arm position).

5″-CAA GCT# TGCCAG TAT CAG ATG CAG-3 'This probe is a 165 sample of C. diversius. with a single unit at position 6 (numbered from the 5' end of the probe strand). It has one T-G mismatch.

Following the procedure described in Example 8, the probe was conjugated to the target rRNA (in this case was hybridized with either C. coli or C. diversillas). profit safety of samples of hybridized and non-hybridized probes; Qualitative properties were tested as described in Example 7 at pH 7.6.

Stability half-life ratio E. Cori 18.6 0.8 23.3 C.D. 1. l*　0-8　　　　　　1.4 Berthius *HAP analysis (as described in Example 2) shows 73% hybridization The lower half-life of ester hydrolysis leads to lower hybridization, as shown in It is not for the purpose of

These data are valid in the case of no% hybridization with perfectly matched targets. However, the AE-probe (as described in previous examples) Hybridization at targets protected from solution but containing a single base mismatch indicates little protection. This is a perfect target versus a single site miss. yielding a 1217-fold difference in the rate of AE-probe hydrolysis between target targets. .

Accordingly, the methods described herein can be used to differentiate between nucleic acid targets containing a single base difference. can be easily identified and distinguished.

Example 10 acridinium ester-internally labeled hydroxide at room temperature under various conditions. Stability of hybridized and non-bridded probes An internally labeled glove (insert, type 1, see example 1) is described in example 8. C. trachomatis rltNAIP Hybridized with g. By the method described in Example 7, various reagents and pH hybridized and non-hybridized probes at room temperature under conditions (see Results). The stability of the buffer was measured.

result: Buffer pH Temperature Hybrid Control Ratio 0.2M borate 7. 6 60℃ 34.13 0.89 38.085% Triton 5% triton 0.2M borate 9.0 Room temperature 468.75 7.7 60 ．． 875% Triton 5% triton 5011IM Phytin 10.0 Room temperature 145.48 (fast) 2 pieces ．． 18 66.73 Acid, O, OS% SD5 398.55 (sl ow) *　　　　　(fast)＊50IIIM Phytin 11.0 Room temperature 175.44 1.31 133.92 Acid, 0.05% SDS *: In the phytic acid system, the kinetics of hydrolysis is 2r51owJ is hydrolysis It means the late phase of the solution.

Based on these data, if a wide range of test conditions can be met, the uniformity guarantee described here can be applied. It is shown that there are various conditions under which the protection test works. Furthermore, HoI7r! i Phytate systems other than salts are also possible, e.g. very high half-life ratios can be obtained. Examples include phytic acid systems at room temperature.

Example 11 Clarification in buffer using probe internally labeled with acridinium ester Detection of dilution series of chlamydia rRNA by the following method. The internally labeled t; probe (similar to Example 2) was hybridized and its reduced the amount of target rRN A (in this case, Chlamydia trachomanis) .

hybridization mixture 0.5. ul probe (12,5 fmol) lμI RNA (10-ζ 1O -3,10-' or 10-'/Jg) 2μm 20% 5DS 1.7μl H,0 4,8μIIMPB% pH6,8 The control mixture was the same as the hybridization mixture except that it contained water instead of rRNA. The reagent blank mixture is the same as the control mixture except that it contains water instead of probe. It was the same as the compound. The mixture was incubated at 60°C for 20 minutes.

After hybridization %I) 0.2M porate of H9 90. Add ul to each sample and incubated at 60°C for 14 minutes. Next, the injector 1 l is 0. 1% H, O, and the pH of injection 2 was 13 instead of 13.2. ．． 8 and described in Example 2 except that the reading time is 7 seconds instead of 10 seconds. The chemiluminescence was read as follows.

result: Reagent blank...116rlu Control...l 24 rlu Hetsudan 10-”pg rRNA-126rlu 2rl*10-’pg rRNA-210rlu 86rlu10-”pg rRNA・ ・・l　　00rlu　　　976rluI　U”pg　rRNA-78 09rlu 7685rlu reagent blank and control represent the average of triplicates; All others represent the average of two replicates.

These results demonstrate that the invention described herein provides a A linear dilution series of target rRNA can be detected up to a sensitivity limit of g;

*How many examples 12 PCR in clinical media using probes internally labeled with acridinium esters. Detection of dilution series of Lamydia rRNA The internally labeled protein was detected by the following method. (as in Example 2) in a clinical sample and its target rRN A (in this case, Chlamydia trachomatis).

50/Jl throat swab (throat 5tab) 6μm 4.8M PB, pH 4,72/71 rRNA (3X 10-', 3XIO-3, 3X lO-' or I; is 3 x 10-1 μg) 2 μl probe (1 pmol) The control mixture was the same as the hybridization mixture except that it contained water instead of rRNA. The reagent blank mixture is the same as the control mixture except that it contains water instead of probe. It was the same as the compound. The mixture was incubated at 60°C for 20 minutes.

After hybridization, 1/3 (20 μl) of each mixture was added to a 0.2 M polymer at a final pH of 9. (after addition of the hybridization mixture to 50 μl of the sample) at 60°C. Incubated for 40 minutes. Next, each sample is described in Example 11. The chemiluminescence was read as follows.

result: Reagent blank...163rlu Control - 6560 rlu λ company style 10-”pg rRNA-8535rlu 1975rlu1 0-copg rRNA-27306rlu 20746rlu10 -'/'grRNA-258194rlu 251634rlu data is 2 Represents the average of multiple measurements.

These data demonstrate that the invention described herein provides approximately 1 A linear dilution series of target rRNA could be detected up to a sensitivity limit of 0'' μg.

Example 13 Bacterial rR in urine using probes internally labeled with acridinium ester Detection of NA dilution series Internally labeled 30mar protein specific for E. coli Lobes (similar to Example 2) were prepared as described above (adenine substitution, type l linker arm). Hybridize the probe in urine by the following method: and decrease the amount of its target rRNA (in this case, E. coli);

hybridization mixture 79.6μl mound 0.4μm 0.25M EDTA, 0.25M EGTA 10μl, 4.8P B, pH 6,8 5μm20%5DS 5 μm probe (0,125 pmol) 1 pk RNA (IO-”, 10- jtV: Hal　O-'μg) except that the control mixture contained water instead of rRNA. is the same as the hybridization mixture, and the reagent blank mixture is used in place of the probe. The mixture was the same as the control mixture except that it contained water. The mixture was incubated at 60°C for 30 minutes. Incubated.

After hybridization, 300 μl of 0.2 M porate, pH 9, was added to each sample. , and incubated for 16 minutes at 60°C. Next, for each sample, m actual examples Chemiluminescence was read as described in 11.

result: Reagent blank...6845rlu Control - 9250 rlu negative control 10-”pg rRNA-9358rlu 8rlu10-2 ．． ug rRNA-44055rlu 4805rlu10”μg rRNA-61800rlu　　　　52550rluResults are the average of two measurements. represent.

Based on these results, the invention described herein provides approximately 5XlO - We were able to detect a linear dilution series of target rRNA up to the sensitivity limit of μg.

Example 14 Internally labeled with acridinium ester I; in clinical media using probe Used in combination with separation process for detection of dilution series of Chlamydia rRMA selective decomposition Internally labeled probes (as in Example 2) were mixed with control and blank. Hybridization in clinical samples (throat slabs) as described in Example 8 containing It became a standard. After hybridization, 1/3 of each sample was removed and as described in Example 8. The sample was subjected to selective decomposition exactly as described above, while 1/3 was removed to the net and stored at room temperature. (i.e., no selective degradation was performed). In other words, selective decomposition is performed. Both the samples tested and the samples that were not tested were compared to the working example except for the following differences: Separate from unhybridized probe using HAP as described in 2: a, with 1 ml of HAP solution (instead of 20Qμ+), b, with 1 ml of HAP solution using cleaning solution (instead of 200 μm), c, washed 3 times (instead of 1 a), d, the final HAP pellet was resuspended in 100 μl of washing solution and its entire The chemiluminescence was measured (as described in the Examples).

result: selective decomposition Negative SOB Positive S2B control (no rRNA) 18 - 4.7 -10-”pg rRNA 27 1.5 10 2.110-2μg rRNA 117 6.5 70 151Q”2g rRNA 933 52 591 1260.332g rRNA 2756 153 1795 373 results were measured twice using the reagent blank that had already been subtracted. Represents the average of fixed values. SOB is the signal to background ratio, i.e. , chemiluminescence at specific rRNA concentrations separated by control chemiluminescence. It is Nessence.

Based on these data, it is possible to achieve high resolution by adding selective degradation before separation. Sensitivity due to low background resulting in signal-to-background ratio improve.

Example 15 Type probes with improved stringency using differential hydrolysis Contains an internal linker arm of “X” (position of insertion indicated by the symbol # below) and labeled with acridinium ester as described in Example 1. to convert and refine This probe is used for Neisseria gonorhoeae (Neisseria gonorrhoeae). rh.

-eae) is exactly complementary to the 165 subunit of However, it is Closely related to N. meningitidis, fruit Cross-reacting under the hybridization stringency conditions cited in Examples 2 and 8. is typically observed. As described in Example 8 (in 200 μl format) This probe was incubated using 0.5 μg of N. Hybridized, 0.2M sodium tetraborate, pH 7.6, 5% Triton Differential hydrolysis (+D , H,) or not subjected to these differential hydrolysis conditions (-D, H,). It was.

The resulting hybrids were separated on magnetic microspheres and purified as described below. Chemiluminescence was measured.

Magnetic amine microspheres (Adv., Cambridge, Massachusetts, USA) BioMag M4100 (BioMag 11400 manufactured by Inst Magnetics) ．． Advanced Magnetics, Inc., Cambridge e, Mass, )) 1 ml of 0.4 MFB at pH 6.0 containing 150 μg. Added. Stir for 10 seconds. Incubate for 5 minutes at 60°C. Stir for 10 seconds Stir. Pace-Mte (registered trademark) magnetic separation rack ( Ken Probe Co., San Diego, California, USA .

Magnetically separate the spheres from the solution using Let go.

Discard liquid. Add 1ml of 0.3M PB with pH 6.0 and stir for 10 seconds. Wash the spheres 3 times by stirring, magnetically separating and discarding the liquid. , repeat washing. 300μl of 0.2M FB at pH 6.0 and 50% Add Lumamide, stir for 10 seconds, incubate for 5 minutes at 60°C, Hybridize by stirring for seconds and then magnetically separating the spheres from the solution. Elute. The solution was poured into a Clinilumat tube. e) and measure the chemiluminescence as described in Example 7.

result: Conditions Chemiluminescence book-D, H, 178,034 +D, H, 748 *: Hybrid signal to control signal expressed in relative fishing units (rlu) (i.e., no rRNA). Precise marks in this format The signal from the target (i.e., N. gonorrhoeae) is typically 7-10XI This is Orlu.

In conclusion, the differential addition as an additional stringency identification step described here Application of water splitting technology greatly reduces signals from undesired cross-reacting sequences In the case of 0 families, N. Gornohoe probe and N. Meningi Tidis cross-reactivity was reduced to over 200 folds. differential hydration The decomposition process reduces the chemiluminescence of the globe when in its non-hybridized form. By constantly reducing (by hydrolysis) increases the susceptibility to unstable hybrids in equilibrium with the lobe, thus This reduces chemiluminescence due to cross-reaction.

Example 16 Chronic myeloid leukemia using an acridinium ester internally labeled probe Detection of chimeric target sequences consistent with disease. A 24 IIler probe containing a car arm (sequence shown above) was carried out. Labeled with AE and purified as described in Example 1: This probe is associated with chronic myeloid leukemia (CML); mon break) and is called the bcr/abl probe. child The chimeric mRNA of 2000 clones the site of the abl gene to another chromosome containing the bcr gene. It is the product of a chimeric gene formed by translocation to the site of

Synthetic NA 60rIlerll representing bcr/abJm cleavage site junction site Target and normal BCR and ABL target in the same site as described in Example 1. The 0I RNA target was synthesized (sequence below) and located near the center of the cleavage site. pG containing a 450 nucleotide fragment of the chimeric bar/abl gene located in Generated by transcription of EM clones. Globe Sense (probe 5e) mRNA transcription of a similar 450 nucleotide fragment in (nse) was produced as a negative control. accomplished.

BCR/ABL probe sequence synthetic target sequence BCR/ABL target ABL target Inserted as above; asterisk (*) indicates link for acridinium ester addition. The insertion position of the anchor arm is shown, and the underlined part shows the abl sequence.

As described in Example 8, approximately 1 pmol of each of the above compounds was added to the bcr/abl glow. hybridize and test these at pH 7.6 as described in Example 7; .

result: Half-life (minutes) Ratio book Target: bar/abl++ RNA 27.7 39.6 bar/ablDNA 15.0 21.4 Control: Pro Bu sense mRNA O,71abl DNA O,71bcrDNA O-71 non-hybridized protein Lobe 　　　　　　　0.7　　-Ne: Exploratory or control half-life/non-high Half-life of hybridized probes These data demonstrate that chimeric bc combined with CML AH designed to connect the cleavage site junction site of r/abl mRNA A transcription Labeled probes are conjugated to the chimeric target and normal sequence by the HPA technique described herein. (and unhybridized probes).

This occurs in the presence of normal non-chimeric nucleic acids, especially chimeric targets (typically genetically diseased This is evidence that can be used to detect

Furthermore, several examples of Mototora have shown that targets other than rRNA (in this case, 1aRNA and DNA A) is resistant to AE hydrolysis when hybridized to an AE-labeled probe. has been shown to provide protection.

fraction number Figure 1 Internally labeled probe, adenine substitution, type 1 minute Figure 2 minutes Figure 3 Internally labeled probe, insert, type 1 minutes Figure 4 Internally labeled probe, insert, type 2 minutes Figure 5 End-labeled probe portion with adjacent probes Figure 6 End-labeled probe portion with adjacent probes Figure 7 End-labeled globe segment with no adjacent probes Figure 8 international search report

Claims (8)

[Claims] 1. a) transport the medium or a portion thereof to (1) the binding partner, the analyte, or the binding partner; A moiety consisting of an analog of an analyte that forms a binding pair, the moiety comprising an analog of an analyte that forms a binding pair. When it becomes a member, it is conjugated with a substance that has a different stability compared to the stability of the unbound form. (2) if said moiety is said analyte or analogue, said moiety; combining the binding partners in a medium; b) selectively degrade the less stable forms of the substance; and c) remaining after the degradation of the substance. characterized by relating the amount of intact material to the presence or amount of analyte in the medium Measuring the presence or amount of an analyte that combines with the binding partner to form a binding pair in a medium How to do it. 2. a) the medium or part thereof when the binding partner becomes a member of the binding pair; When combined with a binding partner conjugated to a substance, the stability differs compared to the stability of the unbound form. , b) selectively decomposes the less stable forms of the substance; c) remaining after the decomposition of the substance; characterized by relating the amount of intact material to the presence or amount of analyte in the medium Measuring the presence or amount of an analyte that combines with the binding partner to form a binding pair in a medium How to do it. 3. a) The medium, or a portion thereof, (1) forms a binding pair with the analyte or binding partner; a moiety consisting of an analog of an analyte that is a member of a binding pair Sometimes, the conjugated moieties and substances have different stability compared to the stability of the unbound form. (2) combining said binding partners; and b) selectively decomposing less stable forms of said substance. , c) the amount of intact material remaining after the decomposition of the material, as determined by the presence or amount of analyte in the medium. with said binding partner in a medium characterized by being associated with said binding partner to form a binding pair. A method of determining the presence or amount of an analyte. 4. a) the medium or a portion thereof is a first binding partner for the analyte, the first binding partner for the analyte; When the binding partner of 1 becomes a member of a binding pair, compared to the stability of the unbound form, (2) a first binding partner conjugated to a substance of differing stability; combining a second binding partner different from the first binding partner; b) selectively decomposes the less stable forms of the substance; c) remaining after the decomposition of the substance; characterized by relating the amount of intact material to the presence or amount of analyte in the medium Measuring the presence or amount of an analyte that combines with the binding partner to form a binding pair in a medium How to do it. 5. a) modify the vehicle or a portion thereof of a nucleotide sequence, and that is substantially complementary thereto; a specific probe, the probe hybridizes to the nucleotide sequence. when conjugated with a substance that differs in stability compared to the stability of the unhybridized form. b) selectively disassembling less stable forms of the shed; c) The amount of intact material remaining after degradation of the material is determined by the presence of nucleotide sequences in the medium or A method for measuring a nucleotide sequence in a medium, the method comprising: 6. a) transport the medium or a portion thereof to (1) the first binding partner or binder for the analyte; A moiety consisting of an analogue of the first binding partner that forms a binding pair that participates in the binding reaction. When the moiety becomes a member of a binding pair, the stability is compared with that of the unbound form. and (2) a moiety conjugated to a substance of different stability with respect to the analyte. combining a second binding partner different from the binding partner; b) selectively decomposes the less stable forms of the substance; c) remaining after the decomposition of the substance; characterized by relating the amount of intact material to the presence or amount of analyte in the medium Measuring the presence or amount of an analyte that combines with the binding partner to form a binding pair in a medium How to do it. 7. According to any one of claims 1 to 6, the substance is an N-acridinium ester. How to put it on. 8. The substance ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [Wherein, X is O, N, S, halogen, substituted phosphorus, substituted sulfur, substituted boron, or substituted Arsenic exchange, Y is O, S or NH, R1 is alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl, substituted Aryl, alkoxy, aryloxy or none when X is halogen, R2 is H, alkyl, alkenyl, aryl, substituted alkenyl, substituted aryl, alkoxy, or, if and only if X is N, aryloxy, R2 is H, amino, hydroxy, thiol, halogen, nitro, amide, acetyl alkyl, alkenyl, aryl, substituted acetyl, substituted alkyl, substituted alkyl Kenyl, substituted aryl, alkoxy or aryloxy, R4 is alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl or Substituted aryl, in which at least one of R1, R2, R3 or R4 is a substituted aryl. Any one of claims 1 to 6 selected from the group consisting of: The method described in.